Method for producing ergosta-5,7-dienol and/or biosynthetic intermediate and/or secondary products thereof in transgenic organisms

ABSTRACT

The present invention relates to a method for the production of ergosta-5,7-dienol and/or its biosynthetic intermediates and/or metabolites by culturing genetically modified organisms, and to the genetically modified organisms, in particular yeasts, themselves.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP2004/002582 filed Mar. 12, 2004 which claims benefit to Germanapplication 103 12 314.8 filed Mar. 19, 2003.

The present invention relates to a method for the production ofergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites by culturing genetically modified organisms, and to thegenetically modified organisms, in particular yeasts, themselves.

Ergosta-5,7-dienol and its biosynthetic intermediates of the sterolmetabolism, such as, for example, farnesol, geraniol, squalene andlanosterol and zymosterol, and its biosynthetic metabolites of thesterol metabolism, for example in mammals, such as, for example,campesterol, pregnenolone, 17-OH-pregnenolone, progesterone,17-OH-progesterone, 11-deoxycortisol, hydrocortisone,deoxycorticosterone or corticosterone, are compounds of high economicalvalue.

Ergosta-5,7-dienol may act as starting compound for the preparation ofsteroid hormones via biotransformations, chemical synthesis orbiotechnological production.

Hydrocortisone has a weak glucocorticoid effect and is a sought-afterstarting compound for the synthesis of active ingredients with a highlyantiinflammatory, abortive or antiproliferative effect.

Squalene is used as building block for the synthesis of terpenes. In itshydrogenated form, it is used as squalane in dermatology and cosmetics,and in its various derivatives as constituent of skincare and haircareproducts.

Other economically utilizable substances are sterols, such as zymosteroland lanosterol, lanosterol being a pivotal raw material and syntheticmaterial for the chemical synthesis of saponins and steroid hormones.Owing to its good skin penetration and spreading properties, lanosterolis used as emulsion auxiliary and active ingredient for skin creams.

An economical method for the production of ergosta-5,7-dienol and/or itsbiosynthetic intermediates and/or metabolites is therefore of greatimportance.

Methods which are particularly economical are biotechnological methodsexploiting natural organisms or organisms optimized by means of geneticmodification which produce ergosta-5,7-dienol and/or its biosyntheticintermediates and/or metabolites.

The genes of the ergosterol metabolism in yeast are largely known andcloned, such as, for example,

nucleic acids encoding an HMG-CoA reductase (HMG) (Bason M. E. et al,(1988) Structural and functional conservation between yeast and human3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limitingenzyme of sterol biosynthesis. Mol Cell Biol 8:3797-3808,

the nucleic acid encoding a truncated HMG-CoA reductase(t-HMG)(Polakowski T, Stahl U, Lang C. (1998) Overexpression of acytosolic hydroxymethylglutaryl-CoA reductase leads to squaleneaccumulation in yeast. Appl Microbiol Biotechnol. January; 49(1):66-71,

the nucleic acid encoding a lanosterol C14-demethylase (ERG11) (Kalb VF, Loper J C, Dey C R, Woods C W, Sutter T R (1986) Isolation of acytochrome P-450 structural gene from Saccharomyces cerevisiae. Gene45(3):237-45,

the nucleic acid encoding a squalene epoxidase (ERG1) (Jandrositz, A.,et al (1991) The gene encoding squalene epoxidase from Saccharomycescerevisiae: cloning and characterization. Gene 107:155-160 and

nucleic acids encoding a squalene synthetase (ERG9) (Jennings, S. M.,(1991): Molecular cloning and characterization of the yeast gene forsqualene synthetase. Proc Natl Acad Sci USA. July 15; 88(14):6038-42).

There are furthermore known processes which aim at increasing thecontent in specific intermediates and catabolites of the sterolmetabolism in yeasts and fungi.

It is known from T. Polakowski, Molekularbiologische Beeinflussung desErgosterolstoffwechsels der Hefe Saccharomyces cerevisiae[Molecular-biological effects on the ergosterol metabolism of the yeastSaccharomyces cerevisiae], Shaker Verlag Aachen, 1999, pages 59 to 66,that increasing the expression rate of HMG-CoA reductase leads to aslightly increased content in early sterols, such as squalene, while thecontent in later sterols, such as ergosterol, does not changesignificantly or even has a tendency to decrease.

Tainaka et al., J, Ferment. Bioeng. 1995, 79, 64-66 furthermore describethat the overexpression of ERG11 (lanosterol C14-demethylase) leads tothe accumulation of 4,4-dimethylzymosterol, but not ergosterol. Incomparison with the wild type, the zymosterol content of thetransformant is increased by a factor of 1.1 to 1.47, depending on thefermentation conditions.

WO 99/16886 describes a method for the production of ergosterol inyeasts which overexpress a combination of the genes tHMG, ERG9, SAT1 andERG1.

EP 486 290 discloses a method for increasing the squalene, zymosterol,ergosta-5,7,-24(28)-trienol and ergosta-5,7-dienol content in yeast byincreasing the HMG-CoA reductase expression rate and simultaneouslyinterrupting the metabolic pathway ofergosta-5,7,24(28)-trienol-22-dehydrogenase, hereinbelow also referredto as Δ22-desaturase (ERG5).

However, the disadvantage of this method is that the ergosta-5,7-dienolyield is still not satisfactory.

It is an object of the present invention to provide a further method forthe production of ergosta-5,7-dienol and/or its biosyntheticintermediates and/or metabolites with advantageous characteristics, suchas a higher product yield.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows vector pUG6 tHMG.

FIG. 2 shows vector pUG6 ERG1.

We have found that this object is achieved by a method for producingergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites in which organisms are cultured which have

a reduced Δ22-desaturase activity and

an increased HMG-CoA reductase activity and

an increased activity of at least one of the activities selected fromthe group consisting of lanosterol C14-demethylase activity, squaleneepoxidase activity and squalene synthetase activity

in comparison with the wild type.

A reduced activity is understood as meaning not only the reduction ofthe activity, but also the complete elimination of the activity.Accordingly, a reduction of an activity also encompasses a quantitativereduction of the relevant protein in the organism through to a completeabsence of the relevant protein, which can be assayed, for example, by alack of detectability of the relevant enzyme activity or a lack ofimmunological detectability of the relevant proteins.

Δ22-desaturase activity is understood as meaning the enzyme activity ofa Δ22-desaturase.

A Δ22-desaturase is understood as meaning a protein with the enzymaticactivity of converting ergosta-5,7-dienol intoergosta-5,7,22,24-tetraen-3β-ol.

Accordingly, Δ22-desaturase activity is understood as meaning the amountof ergosta-5,7-dienol converted, or the amount ofergosta-5,7,22,24-tetraen-3β-ol formed, by the protein Δ22-desaturasewithin a specific period of time.

Thus, in the case of reduced Δ22-desaturase activity in comparison withthe wild type, the amount of ergosta-5,7-dienol converted, or the amountof ergosta-5,7,22,24-tetraen-3β-ol formed, by the protein Δ22-desaturasewithin a specific period of time is reduced in comparison with the wildtype.

The Δ22-desaturase activity is preferably reduced to at least 90%, morepreferably to at least 70%, more preferably to at least 50%, morepreferably to at least 30%, even more preferably by at least 10%, evenmore preferably by at least 5%, in particular to 0% of theΔ22-desaturase activity of the wild type. Especially preferred is,accordingly, the eliminination of the Δ22-desaturase activity in theorganism.

The Δ22-desaturase (ERG5) activity can be determined as describedhereinbelow:

Various concentrations of ergosta-5,7-dienol, isolated from S.cerevisiae Erg5 mutants (Parks et al, 1985. Yeast sterols.yeast mutantsas tools for the study of sterol metabolism. Methods Enzymol.111:333-346) and 50 μg of dilauroylphosphatidylcholin are mixed andsonicated until a white suspension forms. Processed microsomes are added(1 ml)(3 mg/ml protein). NADPH (final concentration 1 mM) is added tothe test mixture in order to start the enzyme reaction. The mixture isincubated for 20 minutes at 37° C. The reaction is stopped by additionof 3 ml of methanol, and sterols are hydrolyzed by addition of 2 ml 60%(wt/vol) KOH in water. The mixture is incubated for 2 hours at 90° C.After cooling, the mixture is extracted three times with 5 ml of hexaneand concentrated by evaporation on a rotary evaporator. The sterols aresubsequently silylated with bis(trimethylsilyl)trifluoroacetamide (50 μlin 50 μl of toluene) for one hour at 60° C. The sterols are analyzed bygas chromatography/mass spectroscopy (GC-MS) (for example Model VG12-250 gas chromatograph-mass spectrometer; VG Biotech, Manchester,United Kingdom). The resulting Δ22-desaturated intermediate can beidentified as a function of the amount of substrate employed. Microsomeswhich are not incubated with substrate act as reference.

This method is a modification of the method described in Lamb et al:Purification, reconstitution, and inhibition of cytochrome P-450 steroldelta22-desaturase from the pathogenic fungus Candida glabrata.Antimicrob Agents Chemother. 1999 July; 43(7):1725-8.

The Δ22-desaturase activity can be reduced independently by differentcytological mechanisms, for example by inhibiting the correspondingactivity at the protein level, for example by addition of inhibitors ofthe enzymes in question, or by reducing the gene expression of thecorresponding nucleic acids encoding a Δ22-desaturase in comparison withthe wild type.

In a preferred embodiment of the method according to the invention, theΔ22-desaturase activity is reduced in comparison with the wild type byreducing the gene expression of the corresponding nucleic acids encodinga Δ22-desaturase.

Reducing the gene expression of the nucleic acids encoding aΔ22-desaturase in comparison with the wild type can likewise be effectedin various ways, for example by

a) introducing nucleic acid sequences which can be transcribed into anantisense nucleic acid sequence which is capable of inhibiting theΔ22-desaturase activity, for example by inhibiting the expression ofendogenous Δ22-desaturase activity,

b) overexpressing homologous Δ22-desaturase nucleic acid sequences,which lead to cosuppression,

c) introducing nonsense mutations into the endogen by introducingRNA/DNA oligonucleotides into the organism,

d) introducing specific DNA-binding factors, for example factors of thezinc finger transcription factor type, which bring about a reduced geneexpression, or

e) generating knock-out mutants, for example with the aid of T-DNAmutagenesis or homologous recombination.

In a preferred embodiment of the method according to the invention, thegene expression of the nucleic acids encoding a Δ22-desaturase isreduced by generating knock-out mutants, especially preferably byhomologous recombination.

Accordingly, it is preferred to use an organism without a functionalΔ22-desaturase gene.

In a preferred embodiment, the generation of knock-out mutants, that isto say the deletion of the target locus Δ22-desaturase gene, is carriedout simultaneously with the integration of an expression cassettecomprising at least one of the nucleic acids described hereinbelow,encoding a protein whose activity is being increased in comparison withthe wild type, by homologous recombination.

To this end, it is possible to use nucleic acid constructs which, inaddition to the expression cassettes described hereinbelow comprisingpromoter, coding sequence and, if appropriate, terminator, and inaddition to a selection marker described hereinbelow, comprise, at the3′ and 5′ end, nucleic acid sequences which are identical to nucleicacid sequences at the beginning and at the end of the gene to bedeleted.

Once selection has taken place, it is preferred to remove the selectionmarker again by means of recombinase systems, for example by loxPsignals at the 3′ and 5′ end of the selection marker, using a Crerecombinase (Cre-LoxP system).

In the preferred organism Saccharomyces cerevisiae, the Δ22-desaturasegene denotes the gene ERG5 (SEQ. ID. NO. 1). SEQ. ID. NO. 2 constitutesthe corresponding Saccharomyces cerevisiae Δ22-desaturase (Skaggs, B. A.et al,: Cloning and characterization of the Saccharomyces cerevisiaeC-22 sterol desaturase gene, encoding a second cytochrome P-450 involvedin ergosterol biosynthesis, Gene. 1996 Feb. 22; 169(1):105-9.).

HMG-CoA reductase activity is understood as meaning the enzyme activityof an HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme Areductase).

An HMG-CoA reductase is understood as meaning a protein with theenzymatic activity of converting 3-hydroxy-3-methylglutaryl-coenzyme Ainto mevalonate.

Accordingly, HMG-CoA reductase activity is understood as meaning theamount of 3-hydroxy-3-methylglutaryl-coenzyme A converted, or the amountof mevalonate formed, by the protein HMG-CoA reductase within a specificperiod of time.

Thus, in the case of an increased HMG-CoA reductase activity incomparison with the wild type, the amount of3-hydroxy-3-methylglutaryl-coenzyme A converted, or the amount ofmevalonate formed, by the protein HMG-CoA reductase within a specificperiod of time is increased in comparison with the wild type.

Preferably, this increase in the HMG-CoA reductase activity amounts toat least 5%, more preferably to at least 20%, more preferably to atleast 50%, more preferably to at least 100%, even more preferably to atleast 300%, especially preferably to at least 500%, in particular to atleast 600% of the HMG-CoA reductase activity of the wild type.

The HMG-CoA reductase activity is determined as described in Th.Polakowski, Molekularbiologische Beeinflussung desErgosterolstoffwechsels der Hefe Saccharomyces cerevisiae,Shaker-Verlag, Aachen 1999, ISBN 3-8265-6211-9.

According to this reference, 10⁹ yeast cells of a 48-hour-old cultureare harvested by centrifugation (3500×g, 5 min) and washed in 2 ml ofbuffer I (100 mM potassium phosphate buffer, pH 7.0). The cell pellet istaken up in 500 μl of buffer 1 (cytosolic proteins) or 2 (100 mMpotassium phosphate buffer pH7.0; 1% Triton X-100) (total proteins), and1 μl of 500 mM PMSF in isopropanol is added. 500 μl of glass beads(d=0.5 mm) are added to the cells, and the cells are disrupted byvortexing 5× for one minute. The liquid between the glass beads istransferred into a fresh Eppendorf tube. Cell debris and membranecomponents are removed by centrifuging for 15 minutes (14000×g). Thesupernatant is transferred into a fresh Eppendorf tube and constitutesthe protein fraction.

The HMG-CoA activity is determined by measuring the consumption ofNADPH+H⁺ in the reduction of 3-hydroxy-3-methylglutaryl-CoA, which isadded as a substrate.

In a reaction volume of 1000 μl there are added 20 μl of yeast proteinisolate together with 910 μl of buffer I; 50 μl of 0.1 M DTT and 10 μlof 16 mM NADPH+H⁺. The reaction mixture is warmed to 30° C. and ismeasured in a photometer for 7.5 minutes at 340 nm. The decrease inNADPH which is measured during this period is the breakdown rate withoutadded substrate and is taken into consideration as background.

Thereafter, substrate is added (10 μl of 30 mM HMG-CoA), and themeasurement is continued for 7.5 minutes. The HMG-CoA reductase activityis calculated by determining the specific NADPH breakdown rate.

Lanosterol C14-demethylase activity is understood as meaning the enzymeactivity of a lanosterol C14-demethylase.

A lanosterol C14-demethylase is understood as meaning a protein with theenzymatic activity of converting lanosterol into4,4-dimethylcholesta-8,14,24-trienol.

Accordingly, lanosterol C14-demethylase activity is understood asmeaning the amount of lanosterol converted, or the amount of4,4-dimethylcholesta-8,14,24-trienol formed, by the protein lanosterolC14-demethylase within a specific period of time.

Thus, in the case of an increased lanosterol C14-demethylase activity incomparison with the wild type, the amount of lanosterol converted, orthe amount of 4,4-dimethylcholesta-8,14,24-trienol formed, by theprotein lanosterol C14-demethylase within a specific period of time isincreased in comparison with the wild type.

Preferably, this increase in the lanosterol C14-demethylase activityamounts to at least 5%, more preferably to at least 20%, more preferablyto at least 50%, more preferably to at least 100%, even more preferablyto at least 300%, especially preferably to at least 500%, in particularto at least 600%, of the lanosterol C14-demethylase activity of the wildtype.

The lanosterol C14-demethylase activity is determined as described inOmura, T and Sato, R. (1964) The carbon monoxide binding pigment inliver microsomes. J. Biol. Chem. 239, 2370-2378. In this test, theamount of P450 enzyme is semiquantifiable as the holoenzyme with boundheme. The (active) holoenzyme (with heme) can be reduced by CO, and onlythe CO-reduced enzyme has an absorption maximum at 450 nm. Thus, theabsorption maximum at 450 nm is a measure for the lanosterolC14-demethylase activity.

To carry out the activity determination, a microsome fraction (4-10mg/ml protein in 100 mM potassium phosphate buffer) is diluted 1:4 insuch a way that the protein concentration employed for the assay is 2mg/ml. The assay is carried out directly in a cell.

A spatula-tipful of dithionite (S₂O₄Na₂) is added to the microsomes. Thebaseline is recorded with a spectrophotometer in the 380-500 nm range.

Approximately 20-30 CO bubbles are subsequently bubbled through thesample. The absorption is now measured in the same range. The absorptionlevel at 450 nm corresponds to the amount of P450 enzyme in the reactionmixture.

Squalene epoxidase activity is understood as meaning the enzyme activityof a squalene epoxidase.

A squalene epoxidase is understood as meaning a protein with theenzymatic activity of converting squalene into squalene epoxide.

Accordingly, squalene epoxidase activity is understood as meaning theamount of squalene converted, or the amount of squalene epoxide formed,by the protein squalene epoxidase within a specific period of time.

Thus, in the case of an increased squalene epoxidase activity incomparison with the wild type, the amount of squalene converted, or theamount of squalene epoxide formed, by the protein squalene epoxidasewithin a specific period of time is increased in comparison with thewild type.

Preferably, this increase in squalene epoxidase activity amounts to atleast 5%, more preferably to at least 20%, more preferably to at least50%, more preferably to at least 100%, even more preferably to at least300%, especially preferably to at least 500%, in particular to at least600% of the squalene epoxidase activity of the wild type.

The squalene epoxidase activity is determined as described in Leber R,Landl K, Zinser E, Ahorn H, Spok A, Kohlwein S D, Turnowsky F, Daum G.(1998) Dual localization of squalene epoxidase, Erg1p, in yeast reflectsa relationship between the endoplasmic reticulum and lipid particles,Mol. Biol. Cell. 1998, February; 9(2):375-86.

This method comprises 0.35 to 0.7 mg of microsomal protein or 3.5 to 75μg of lipid particle protein in 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1mM FAD, 3 mM NADPH, 0.1 mM squalene 2,3-epoxidase cyclase inhibitorU18666A, 32 μM [³H]squalene dispersed in 0.005% Tween 80 in a totalvolume of 500 μl.

The assay is carried out at 30° C. After pretreatment for 10 minutes,the reaction is started by addition of squalene and stopped after 15, 30or 45 minutes by lipid extraction with 3 ml of chloroform/methanol (2:1vol/vol) and 750 μl 0.035% MgCl₂.

The lipids are dried under nitrogen and redissolved in 0.5 ml ofchloroform/methanol (2:1 vol/vol). For a thin-layer chromatography,portions are placed on a silica gel 60 plate (0.2 mm) and separated withchloroform as the eluant. The positions containing [³H]2,3-oxidosqualeneand [³H]squalene were scraped out and quantified with a scintillationcounter.

Squalene synthetase activity is understood as meaning the enzymeactivity of a squalene synthetase.

Squalene synthetase is understood as meaning a protein with theenzymatic activity of converting farnesyl pyrophosphate into squalene.

Accordingly, squalene synthetase activity is understood as meaning theamount of farnesyl pyrophosphate converted, or the amount of squaleneformed, by the protein squalene synthetase within a specific period oftime.

Thus, in the case of an increased squalene synthetase activity incomparison with the wild type, the amount of farnesyl pyrophosphateconverted, or the amount of squalene formed, by the protein squalenesynthetase within a specific period of time is increased in comparisonwith the wild type.

Preferably, this increase in squalene synthetase activity amounts to atleast 5%, more preferably to at least 20%, more preferably to at least50%, more preferably to at least 100%, even more preferably to at least300%, especially preferably to at least 500%, in particular to at least600% of the squalene synthetase activity of the wild type.

The squalene synthetase activity can be determined as describedhereinbelow:

The reaction mixtures comprise 50 mM Mops, pH 7.2, 10 mM MgCl₂, 1% (v/v)Tween-80, 10% (v/v) 2-propanol, 1 mM DTT, 1 mg/ml BSA, NADPH, FPP (orPSPP) and microsomes (protein content 3 mg) in a total volume of 200 μlin glass tubes. Reactions with radioactive substrate [1-³H]FPP (15-30mCi/μmol) are incubated for 30 minutes at 30° C., and the suspensionmixture is filled up with 1 volume of 1:1 (v/v) 40% aqueousKOH:methanol. Liquid NaCl is added until the solution is saturated, and2 ml of ligroin comprising 0.5% (v/v) squalene are likewise added.

The suspension is vortexed for 30 seconds. Using a Pasteur pipette, 1 mlportions of the ligroin layer are applied to a packed 0.5×6 cm aluminumcolumn (80-200 mesh, Fisher). The column is preequilibrated with 2 ml ofligroin comprising 0.5% (v/v) squalene. The column is subsequentlyeluted with 5×1 ml toluene comprising 0.5% (v/v) squalene. The squaleneradioactivity is measured in Cytoscint (ICN) scintillation cocktailusing a scintillation counter (Beckman).

This method is a modification of the methods described in Radisky etal., Biochemistry. 2000 Feb. 22; 39(7):1748-60, Zhang et al. (1993)Arch. Biochem. Biophys. 304, 133-143 and Poulter, C. D. et al. (1989) J.Am. Chem. Soc. 111, 3734-3739.

A wild type is understood as meaning the correspondingnon-genetically-modified starting organism. Preferably, and inparticular in cases where the organism or the wild type are notunambiguously identifiable, the wild type for the reduction of theΔ22-desaturase activity, the increase in the HMG-CoA reductase activity,the increase in the lanosterol C14-demethylase activity, the increase inthe squalene epoxidase activity and the increase in the squalenesynthetase activity, and for the increase in the content inergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites is understood as meaning a reference organism. Thisreference organism is preferably the yeast strain Saccharomycescerevisiae AH22.

The HMG-CoA reductase activity, the lanosterol C14-demethylase activity,the squalene epoxidase activity or the squalene synthetase activity canbe increased independently in various ways, for example by eliminatinginhibiting regulatory mechamisms at the expression and protein level, orby increasing the gene expression of the corresponding nucleic acids,that is to say nucleic acids encoding an HMG-CoA reductase, lanosterolC14-demethylase, squalene epoxidase or squalene synthetase, incomparison with the wild type.

Increasing the gene expression of the corresponding nucleic acid incomparison with the wild type can likewise be effected in various ways,for example by inducing the corresponding genes by activators, that isto say by inducing the HMG-CoA reductase gene, the lanosterolC14-demethylase gene, the squalene epoxidase gene or the squalenesynthetase gene by activators or by introducing one or more gene copiesof the corresponding nucleic acids, that is to say by introducing, intothe organism, one or more nucleic acids encoding an HMG-CoA reductase,lanosterol C14-demethylase, squalene epoxidase or squalene synthetase.

In accordance with the invention, increasing the gene expression of anucleic acid encoding an HMG-CoA reductase, lanosterol C14-demethylase,squalene epoxidase or squalene synthetase is also understood as meaningthe manipulation of the expression of the organism's own, in particularthe yeast's own, endogenous HMG-CoA reductases, lanosterolC14-demethylases, squalene epoxidases or squalene synthetases.

This can be achieved for example by modifying the promoter DNA sequencefor genes encoding HMG-CoA reductase, lanosterol C14-demethylase,squalene epoxidase or squalene synthetase. Such a modification, whichresults in an increased expression rate of the gene in question, can bebrought about for example by deletion or insertion of DNA sequences.

As described above, it is possible to modify the expression of theendogenous HMG-CoA reductase, lanosterol C14-demethylase, squaleneepoxidase or squalene synthetase by applying exogenous stimuli. This canbe brought about by specific physiological conditions, that is to say bythe application of foreign substances.

Moreover, a modified or increased expression of endogenous HMG-CoAreductase, lanosterol C14-demethylase, squalene epoxidase or squalenesynthetase genes can be achieved by a regulator protein which does notoccur in the nontransformed organism interacts with the promoter ofthese genes.

Such a regulator may be a chimeric protein consisting of a DNA bindingdomain and a transcription activator domain, as described, for example,in WO 96/06166.

In a preferred embodiment, the increase in lanosterol C14-demethylaseactivity in comparison with the wild type is effected by increasing thegene expression of a nucleic acid encoding a lanosterol C14-demethylase.

In a furthermore preferred embodiment, the increase in the geneexpression of a nucleic acid encoding a lanosterol C14-demethylase iseffected by introducing, into the organism, one or more nucleic acidsencoding a lanosterol C14-demethylase.

In principle, any lanosterol C14-demethylase gene (ERG11), that is tosay any nucleic acid encoding a lanosterol C14-demethylase, may be usedfor this purpose. In the case of genomic lanosterol C14-demethylasenucleic acid sequences from eukaryotic sources, which contain introns,and in the event that the host organism is not capable, or cannot bemade capable, of expressing the corresponding lanosterolC14-demethylase, it is preferred to use preprocessed nucleic acidsequences, such as the corresponding cDNAs.

Examples of lanosterol C14-demethylase genes are nucleic acids encodinga lanosterol C14-demethylase from Saccharomyces cerevisiae (Kalb V F,Loper J C, Dey C R, Woods C W, Sutter T R (1986) Isolation of acytochrome P-450 structural gene from Saccharomyces cerevisiae. Gene45(3):237-45), Candida albicans (Lamb D C, Kelly D E, Baldwin B C, GozzoF, Boscott P, Richards W G, Kelly S L (1997) Differential inhibition ofCandida albicans CYP51 with azole antifungal stereoisomers. FEMSMicrobiol Lett 149(1):25-30), Homo sapiens (Stromstedt M, Rozman D,Waterman M R. (1996) The ubiquitously expressed human CYP51 encodeslanosterol 14 alpha-demethylase, a cytochrome P450 whose expression isregulated by oxysterols. Arch Biochem Biophys 1996 May 1; 329(1):73-81c)or Rattus norvegicus, Aoyama Y, Funae Y, Noshiro M, Horiuchi T, YoshidaY. (1994) Occurrence of a P450 showing high homology to yeast lanosterol14-demethylase (P450(14DM)) in the rat liver. Biochem Biophys ResCommun. June 30; 201(3):1320-6).

In the transgenic organisms according to the invention, there thusexists, in this preferred embodiment, at least one further lanosterolC14-demethylase gene in comparison with the wild type.

The number of the lanosterol C14-demethylase genes in the transgenicorganisms according to the invention is at least two, preferably morethan two, especially preferably more than three, very especiallypreferably more than five.

All of the nucleic acids mentioned in the description may be, forexample, an RNA, DNA or cDNA sequence.

The above-described method preferably employs nucleic acids encodingproteins comprising the amino acid sequence SEQ. ID. NO. 6 or a sequencederived from this sequence by substitution, insertion or deletion ofamino acids which has at least 30%, preferably at least 50%, morepreferably at least 70%, especially preferably at least 90%, mostpreferably at least 95% identity with the sequence SEQ. ID. NO. 6 at theamino acid level, which proteins have the enzymatic characteristic of alanosterol C14-demethylase.

The sequence SEQ. ID. NO. 6 constitutes the amino acid sequence of theSaccharomyces cerevisiae lanosterol C14-demethylase.

Further examples of lanosterol C14-demethylases and lanosterolC14-demethylase genes can be found readily, for example from variousorganisms whose genomic sequence is known, by homology comparisons ofthe amino acid sequences or of the corresponding backtranslated nucleicacid sequences from databases with SEQ. ID. NO. 2.

Further examples of lanosterol C14-demethylases and lanosterolC14-demethylase genes can be found readily in a manner known per se byhybridization and PCR techniques from various organisms whose genomicsequence is not known, for example starting from the sequence SEQ. ID.NO. 5.

In the description, the term “substitution” is understood as meaning thesubstitution of one or more amino acids by one or more amino acids. Itis preferred to perform what are known as conservative substitutions, inwhich the replacement amino acid has a similar property to the originalamino acid, for example the substitution of Glu by Asp, Gln by Asn, Valby Ile, Leu by Ile, Ser by Thr.

Deletion is the replacement of an amino acid by a direct bond. Preferredpositions for deletions are the termini of the polypeptide and thelinkages between the individual protein domains.

Insertions are introductions of amino acids into the polypeptide chain,a direct bond formally being replaced by one or more amino acids.

Identity between two proteins is understood as meaning the identity ofthe amino acids over in each case the entire protein length, inparticular the identity calculated by alignment with the aid of theLasergene software from DNASTAR, inc. Madison, Wis. (USA) using theClustal method (Higgins D G, Sharp P M. Fast and sensitive multiplesequence alignments on a microcomputer. Comput Appl. Biosci. 1989 April;5(2):151-1), setting the following parameters:

-   Multiple alignment parameter:-   Gap penalty 10-   Gap length penalty 10-   Pairwise alignment parameter:-   K-tuple 1-   Gap penalty 3-   Window 5-   Diagonals saved 5

Accordingly, a protein with an identity of at least 30% with thesequence SEQ. ID. NO. 6 at the amino acid level is understood as meaninga protein which has at least 30% identity when its sequence is alignedwith the sequence SEQ. ID. NO. 6, in particular in accordance with theabove program algorithm with the above parameter set.

In a furthermore preferred embodiment, nucleic acids encoding proteinscomprising the amino acid sequence of the Saccharomyces cerevisiaelanosterol C14-demethylase (SEQ. ID. NO. 6) are introduced intoorganisms.

Suitable nucleic acid sequences can be obtained for example bybacktranslating the polypeptide sequence in accordance with the geneticcode.

Codons which are preferably used for this purpose are those which areused frequently in accordance with the organism-specific codon usage.The codon usage can be determined readily with the aid of computerevaluations of other, known genes of the organisms in question.

If, for example, the protein is to be expressed in yeast, it isfrequently advantageous to use the yeast codon usage whenbacktranslating.

In an especially preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 5 is introduced into the organism.

The sequence SEQ. ID. NO. 5 constitutes the genomic DNA fromSaccharomyces cerevisiae (ORF S0001049), which encodes the lanosterolC14-demethylase with the sequence SEQ ID NO. 6.

All of the abovementioned lanosterol C14-demethylase genes canfurthermore be generated from the nucleotide units by chemical synthesisin a manner known per se, such as, for example, by fragment condensationof individual overlapping complementary nucleic acid units of the doublehelix. Oligonucleotides can be synthesized chemically for example in aknown manner using the phosphoamidite method (Voet, Voet, 2nd Edition,Wiley Press New York, pages 896-897). The annealment of syntheticoligonucleotides and the filling in of gaps with the aid of the DNApolymerase Klenow fragment and ligation reactions are described inSambrook et al. (1989), Molecular cloning: A laboratory manual, ColdSpring Harbor Laboratory Press, as are general cloning methods.

In a preferred embodiment, increasing the HMG-CoA reductase activity incomparison with the wild type is effected by increasing the geneexpression of a nucleic acid encoding an HMG-CoA reductase.

In an especially preferred embodiment of the method according to theinvention, increasing the gene expression of a nucleic acid encoding anHMG-CoA reductase is effected by introducing, into the organism, anucleic acid construct comprising a nucleic acid encoding an HMG-CoAreductase whose expression in the organism is subject to reducedregulation in comparison with the wild type.

Reduced regulation in comparison with the wild type is understood asmeaning a regulation which is reduced in comparison with theabove-defined wild type, preferably no regulation, at the expression orprotein level.

The reduced regulation can preferably be achieved by means of a promoterwhich is operably linked to the coding sequence in the nucleic acidconstruct and which, in the organism, is subject to reduced regulationin comparison with the wild-type promoter.

For example, the middle ADH promoter in yeast is only subject to reducedregulation and is therefore particularly preferred as promoter in theabove-described nucleic acid construct.

This promoter fragment of the ADH12s promoter, hereinbelow also referredto as ADH1, shows almost constitutive expression (Ruohonen L, PenttilaM, Keranen S. (1991) Optimization of Bacillus alpha-amylase productionby Saccharomyces cerevisiae. Yeast. May-June; 7(4):337-462; Lang C,Looman A C. (1995) Efficient expression and secretion of Aspergillusniger RH5344 polygalacturonase in Saccharomyces cerevisiae. ApplMicrobiol Biotechnol. December; 44(1-2):147-56.), so that thetranscriptional regulation no longer proceeds via ergosterolbiosynthesis intermediates.

Further preferred promoters with reduced regulation are constitutivepromoters such as, for example, the yeast TEF1 promoter, the yeast GPDpromoter or the yeast PGK promoter (Mumberg D, Muller R, Funk M. (1995)Yeast vectors for the controlled expression of heterologous proteins indifferent genetic backgrounds. Gene. 1995 Apr. 14; 156(1):119-22; Chen CY, Oppermann H, Hitzeman R A. (1984) Homologous versus heterologous geneexpression in the yeast, Saccharomyces cerevisiae. Nucleic Acids Res.December 11; 12(23):8951-70.).

In a further preferred embodiment, reduced regulation can be achieved byusing, as the nucleic acid encoding an HMG-CoA reductase, a nucleic acidwhose expression in the organism is subject to reduced regulation incomparison with the homologous, orthologous nucleic acid.

It is especially preferred to use a nucleic acid which only encodes thecatalytic region of the HMG-CoA reductase (truncated (t-)HMG-CoAreductase) as the nucleic acid encoding an HMG-CoA reductase. Thisnucleic acid (t-HMG), which is described in EP 486 290 and WO 99/16886,only encodes the catalytically active portion of the HMG-CoA reductasewhile the membrane domain, which is responsible for the regulation atthe protein level, is absent. Thus, this nucleic acid is subjected toreduced regulation, in particular in yeast, and leads to an increasedgene expression of the HMG-CoA reductase.

The above-described nucleic acid construct can be incorporated into thehost organism either chromosomally using integration vectors orepisomally using episomal plasmids, in each case comprising theabove-described nucleic acid construct.

In an especially preferred embodiment, nucleic acids are introduced,preferably via the above-described nucleic acid construct, which encodeproteins comprising the amino acid sequence SEQ. ID. NO. 4 or a sequencederived from this sequence by substitution, insertion or deletion ofamino acids which has at least 30% identity with the sequence SEQ. ID.NO. 4 at the amino acid level, which proteins have the enzymaticcharacteristic of an HMG-CoA reductase.

The sequence SEQ. ID. NO. 4 constitutes the amino acid sequence of thetruncated HMG-CoA reductase (t-HMG).

Further examples of HMG-CoA reductases, and thus also of the t-HMG-CoAreductases which are reduced to the catalytic portion, or the codinggenes, can be found readily, for example from various organisms whosegenomic sequence is known, by homology comparisons of the amino acidsequences or of the corresponding back-translated nucleic acid sequencesfrom databases with SEQ ID. NO. 4.

Further examples of HMG-CoA reductases, and thus also of the t-HMG-CoAreductases which are reduced to the catalytic portion, or the codinggenes, can be found readily from various organisms whose genomicsequence is not known by hybridization and PCR techniques in a mannerknown per se, for example starting from the sequence SEQ. ID. No. 3.

It is especially preferred to use a nucleic acid comprising the sequenceSEQ. ID. NO. 3 as nucleic acid encoding a truncated HMG-CoA reductase.

In an especially preferred embodiment, the reduced regulation isachieved by using, as nucleic acid encoding an HMG-CoA reductase, anucleic acid whose expression in the organism is subject to reducedregulation in comparison with the organism's own, orthologous nucleicacid and by using a promoter which is subject to reduced regulation inthe organism in comparison with the wild-type promoter.

In a preferred embodiment, increasing the squalene epoxidase activity incomparison with the wild type is effected by increasing the geneexpression of a nucleic acid encoding a squalene epoxidase.

In a furthermore preferred embodiment, increasing the gene expression ofa nucleic acid encoding a squalene epoxidase is effected by introducing,into the organism, one or more nucleic acids encoding squaleneepoxidase.

In principle, any squalene epoxidase gene (ERG1), that is to say anynucleic acid which encodes a squalene epoxidase, may be used for thispurpose. In the case of genomic squalene epoxidase nucleic acidsequences from eukaryotic sources, which contain introns, and in theevent that the host organism is not capable, or cannot be made capable,of expressing the corresponding squalene epoxidase, it is preferred touse preprocessed nucleic acid sequences, such as the correspondingcDNAs.

Examples of nucleic acids encoding a squalene epoxidase are nucleicacids encoding a squalene epoxidase from Saccharomyces cerevisiae(Jandrositz, A., et al (1991) The gene encoding squalene epoxidase fromSaccharomyces cerevisiae: cloning and characterization. Gene107:155-160, from Mus musculus (Kosuga K, Hata S, Osumi T, Sakakibara J,Ono T. (1995) Nucleotide sequence of a cDNA for mouse squaleneepoxidase, Biochim Biophys Acta, February 21; 1260(3):345-8b), fromRattus norvegicus (Sakakibara J, Watanabe R, Kanai Y, Ono T. (1995)Molecular cloning and expression of rat squalene epoxidase. J Biol ChemJanuary 6; 270(1):17-20c) or from Homo sapiens (Nakamura Y, SakakibaraJ, Izumi T, Shibata A, Ono T. (1996) Transcriptional regulation ofsqualene epoxidase by sterols and inhibitors in HeLa cells., J. Biol.Chem. 1996, April 5; 271(14):8053-6).

In the transgenic organisms according to the invention, there thusexists, in this preferred embodiment, at least one further squaleneepoxidase gene in comparison with the wild type.

The number of the squalene epoxidase genes in the transgenic organismsaccording to the invention is at least two, preferably more than two,especially preferably more than three, very especially preferably morethan five.

The above-described method preferably employs nucleic acids encodingproteins comprising the amino acid sequence SEQ. ID. NO. 8 or a sequencederived from this sequence by substitution, insertion or deletion ofamino acids which has at least 30%, preferably at least 50%, morepreferably at least 70%, especially preferably at least 90%, mostpreferably at least 95% identity with the sequence SEQ. ID. NO. 8 at theamino acid level, which proteins have the enzymatic characteristic of asqualene epoxidase.

The sequence SEQ. ID. NO. 8 constitutes the amino acid sequence of theSaccharomyces cerevisiae squalene epoxidase.

Further examples of squalene epoxidases and squalene epoxidase genes canbe found readily, for example from various organisms whose genomicsequence is known, by homology comparisons of the amino acid sequencesor of the corresponding backtranslated nucleic acid sequences fromdatabases with SEQ. ID. NO. 8.

Further examples of squalene epoxidase and squalene epoxidase genes canbe found readily in a manner known per se by hybridization and PCRtechniques from various organisms whose genomic sequence is not known,for example starting from the sequence SEQ. ID. NO. 7.

In a furthermore preferred embodiment, nucleic acids encoding proteinscomprising the amino acid sequence of the Saccharomyces cerevisiaesqualene epoxidase (SEQ. ID. NO. 8) are introduced into organisms.

Suitable nucleic acid sequences can be obtained for example bybacktranslating the polypeptide sequence in accordance with the geneticcode.

Codons which are preferably used for this purpose are those which areused frequently in accordance with the organism-specific codon usage.The codon usage can be determined readily with the aid of computerevaluations of other, known genes of the organisms in question.

If, for example, the protein is to be expressed in yeast, it isfrequently advantageous to use the yeast codon usage whenbacktranslating.

In an especially preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 7 is introduced into the organism.

The sequence SEQ. ID. NO. 7 constitutes the genomic DNA fromSaccharomyces cerevisiae (ORF S0003407), which encodes the squaleneepoxidase with the sequence SEQ ID NO. 8.

All of the abovementioned squalene epoxidase genes can furthermore begenerated from the nucleotide units by chemical synthesis in a mannerknown per se, such as, for example, by fragment condensation ofindividual overlapping complementary nucleic acid units of the doublehelix. Oligonucleotides can be synthesized chemically for example in aknown manner using the phosphoamidite method (Voet, Voet, 2nd Edition,Wiley Press New York, pages 896-897). The annealment of syntheticoligonucleotides and the filling in of gaps with the aid of the DNApolymerase Klenow fragment and ligation reactions are described inSambrook et al. (1989), Molecular cloning: A laboratory manual, ColdSpring Harbor Laboratory Press, as are general cloning methods.

In a preferred embodiment, increasing the squalene synthetase activityin comparison with the wild type is effected by increasing the geneexpression of a nucleic acid encoding a squalene synthetase.

In a furthermore preferred embodiment, increasing the gene expression ofa nucleic acid encoding a squalene synthetase is effected byintroducing, into the organism, one or more nucleic acids encoding asqualene synthetase.

In principle, any squalene synthetase gene (ERG9), that is to say anynucleic acid which encodes a squalene synthetase, may be used for thispurpose. In the case of genomic squalene synthetase nucleic acidsequences from eukaryotic sources, which contain introns, and in theevent that the host organism is not capable, or cannot be made capable,of expressing the corresponding squalene synthetase, it is preferred touse preprocessed nucleic acid sequences, such as the correspondingcDNAs.

Examples of nucleic acids encoding a squalene synthetase are nucleicacids encoding a squalene synthetase from Saccharomyces cerevisiae(ERG9), (Jennings, S. M., (1991): Molecular cloning and characterizationof the yeast gene for squalene synthetase. Proc Natl Acad Sci USA. July15; 88(14):6038-42), nucleic acids encoding a squalene synthetase fromBotryococcus braunii Okada (Devarenne, T. P. et al.: Molecularcharacterization of squalene synthase from the green microalgaBotryococcus braunii, raceB, Arch. Biochem. Biophys. 2000, Jan. 15,373(2):307-17), nucleic acids encoding a squalene synthetase from potatotuber (Yoshioka H. et al.: cDNA cloning of sesquiter penecyclase andsqualene synthase, and expression of the genes in potato tuber infectedwith Phytophthora infestans, Plant. Cell. Physiol. 1999, September;40(9):993-8) or nucleic acids encoding a squalene synthetase fromGlycyrrhiza glabra (Hayashi, H. et al.: Molecular cloning andcharacterization of two cDNAs for Glycyrrhiza glabra squalene synthase,Biol. Pharm. Bull. 1999, September; 22(9):947-50.

In the transgenic organisms according to the invention, there thusexists, in this preferred embodiment, at least one further squalenesynthetase gene in comparison with the wild type.

The number of the squalene synthetase genes in the transgenic organismsaccording to the invention is at least two, preferably more than two,especially preferably more than three, very especially preferably morethan five.

The above-described method preferably employs nucleic acids encodingproteins comprising the amino acid sequence SEQ. ID. NO. 10 or asequence derived from this sequence by substitution, insertion ordeletion of amino acids which has at least 30%, preferably at least 50%,more preferably at least 70%, especially preferably at least 90%, mostpreferably at least 95% identity with the sequence SEQ. ID. NO. 10 atthe amino acid level, which proteins have the enzymatic characteristicof a squalene synthetase.

The sequence SEQ. ID. NO. 10 constitutes the amino acid sequence of theSaccharomyces cerevisiae squalene synthetase (ERG9).

Further examples of squalene synthetases and squalene synthetase genescan be found readily, for example from various organisms whose genomicsequence is known, by homology comparisons of the amino acid sequencesor of the corresponding backtranslated nucleic acid sequences fromdatabases with SEQ. ID. NO. 10.

Further examples of squalene synthetases and squalene synthetase genescan be found readily in a manner known per se by hybridization and PCRtechniques from various organisms whose genomic sequence is not known,for example starting from the sequence SEQ. ID. NO. 9.

In a furthermore preferred embodiment, nucleic acids encoding proteinscomprising the amino acid sequence of the Saccharomyces cerevisiaesqualene synthetase (SEQ. ID. NO. 10) are introduced into organisms.

Suitable nucleic acid sequences can be obtained for example bybacktranslating the polypeptide sequence in accordance with the geneticcode.

Codons which are preferably used for this prupose are those which areused frequently in accordance with the organism-specific codon usage.The codon usage can be determined readily with the aid of computerevaluations of other, known genes of the organisms in question.

If, for example, the protein is to be expressed in yeast, it isfrequently advantageous to use the codon usage of yeast whenbacktranslating.

In an especially preferred embodiment, a nucleic acid comprising thesequence SEQ. ID. NO. 9 is introduced into the organism.

The sequence SEQ. ID. NO. 9 constitutes the genomic DNA fromSaccharomyces cerevisiae (ORF YHR190W), which encodes the squalenesynthetase of the sequence SEQ. ID. NO. 10.

All of the abovementioned squalene synthetase genes can furthermore begenerated from the nucleotide units by chemical synthesis in a mannerknown per se, such as, for example, by fragment condensation ofindividual overlapping complementary nucleic acid units of the doublehelix. Oligonucleotides can be synthesized chemically for example in aknown manner using the phosphoamidite method (Voet, Voet, 2nd Edition,Wiley Press New York, pages 896-897). The annealment of syntheticoligonucleotides and the filling in of gaps with the aid of the DNApolymerase Klenow fragment and ligation reactions are described inSambrook et al. (1989), Molecular cloning: A laboratory manual, ColdSpring Harbor Laboratory Press, as are general cloning methods.

The organisms cultured in the method according to the invention areorganisms which have a reduced Δ22-desaturase activity and an increasedHMG-CoA reductase activity and an increased activity of at least one ofthe activities selected from the group consisting of lanosterolC14-demethylase activity, squalene epoxidase activity and squalenesynthetase activity in comparison with the wild type.

In a preferred embodiment, the organisms cultured are organisms whichhave a reduced Δ22-desaturase activity and an increased HMG-CoAreductase activity and an increased lanosterol C14-demethylase activity,squalene epoxidase activity or squalene synthetase activity incomparison with the wild type.

In an especially preferred embodiment of the method according to theinvention, the organisms have a reduced Δ22-desaturase activity and anincreased HMG-CoA reductase activity and an increased activity of atleast two of the activities selected from the group consisting oflanosterol C14-demethylase activity, squalene epoxidase activity andsqualene synthetase activity in comparison with the wild type.

Especially preferred combinations are a reduced Δ22-desaturase activityand an increased HMG-CoA reductase activity and an increased lanosterolC14-demethylase activity and squalene epoxidase activity or lanosterolC14-demethylase activity and squalene synthetase activity or anincreased squalene epoxidase activity and squalene synthetase activityin comparison with the wild type.

In a very especially preferred embodiment of the method according to theinvention, the organisms have a reduced Δ22-desaturase activity and anincreased HMG-CoA reductase activity and an increased lanosterolC14-demethylase activity and an increased squalene epoxidase activityand an increased squalene synthetase activity in comparison with thewild type.

Organisms or genetically modified organisms are understood as meaning,in accordance with the invention, for example bacteria, in particularbacteria of the genus Bacillus, Escherichia coli, Lactobacillus spec. orStreptomyces spec.,

for example yeasts, in particular yeasts of the genus Saccharomycescerevisiae, Pichia pastoris or Klyveromyces spec.,

for example fungi, in particular fungi of the genus Aspergillus spec.,Penicillium spec. or Dictyostelium spec.,

and, for example, also insect cell lines which are capable of generatingergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites, either as the wild type or owing to preceding geneticmodification.

Especially preferred organisms or genetically modified organisms areyeasts, in particular of the species Saccharomyces cerevisiae, inparticular the yeast strains Saccharomyces cerevisiae AH22,Saccharomyces cerevisiae GRF, Saccharomyces cerevisiae DBY747 andSaccharomyces cerevisiae BY4741.

The biosynthetic intermediates of ergosta-5,7-dienol are understood asmeaning all those compounds which occur as intermediates in theergosta-5,7-dienol biosynthesis in the organism used, preferably thecompounds mevalonate, farnesyl pyrophosphate, geraniol pyrophosphate,squalene epoxide, 4-dimethylcholesta-8,14,24-trienol,4,4-dimethylzymosterol, squalene, farnesol, geraniol, lanosterol,zymosterone and zymosterol.

The biosynthetic metabolites of ergosta-5,7-dienol are understood asmeaning all those compounds which are biosynthetic derivatives ofergosta-5,7-dienol in the organism used, that is to say in whichergosta-5,7-dienol occurs as intermediate. They may be compounds whichthe organism used produces naturally from ergosta-5,7-dienol.

However, they are also understood as meaning compounds which can only beproduced from ergosta-5,7-dienol in the organism by introducing genesand enzyme activities from other organisms to which the startingorganism has no orthologous gene.

Owing to the introduction of further plant genes and/or mammalian genesinto yeast it is possible, for example, to produce biosyntheticergosta-5,7-dienol metabolites which only occur naturally in plantsand/or mammals in this yeast.

The introduction into yeast of, for example, nucleic acids encoding aplant Δ7-reductase (DWF5) or its functional equivalents or variants andof nucleic acids encoding mature forms of CYP11A1, ADX(FDX1), ADR (FDXR)and 3β-HSD or their functional equivalents or variants leads to thebiosynthesis of progesterone in this yeast. A detailed description ofthe procedure and of the methods and materials for the correspondinggenetic modification of yeast is published in C. Duport et al., Nat.Biotechnol. 1998, 16, 186-189 and in the references cited therein, whichare herewith expressly incorporated by reference.

The introduction into yeast of, for example, nucleic acids encoding aplant Δ7-reductase (DWF5) or its functional equivalents or variants andof nucleic acids encoding mature forms of CYP11A1, ADX(FDX1) and ADR(FDXR) or their functional equivalents or variants and of nucleic acidsencoding mitochondrial forms of ADX and CYP11B1, 3b-HSD, CYP17A1 andCYP21A1 or their functional equivalents or variants leads to thebiosynthesis of hydrocortisone, 11-deoxycortisol, corticosterone andacetalpregnenolone.

To further increase the content in biosynthetic ergosta-5,7-dienolmetabolites such as, for example, hydrocortisone, it is additionallyadvantageous to suppress wasteful metabolic pathways, that is to saybiosynthetic pathways which do not lead to the desired product. Forexample, the reduction of the activities of the gene products of ATF2,GCY1 and YPR1, especially preferably the deletion of these activities,in yeast leads to a further increase in the hydrocortisone content.

A detailed description of this procedure and of the methods andmaterials for the corresponding genetic modification of yeast ispublished in F. M. Szczebara et al., Nat. Biotechnol. 2003, 21, 143-149and in the references cited therein, which are herewith expresslyincorporated by reference.

The biosynthetic ergosta-5,7-dienol metabolites are therefore understoodas meaning in particular campesterol, pregnenolone, 17-OH pregnenolone,progesterone, 17-OH-progesterone, 11-deoxycortisol, hydrocortisone,deoxycorticosterone and/or corticosterone.

Preferred biosynthetic metabolites are progesterone, corticosterone andhydrocortisone, especially preferably hydrocortisone.

Some of the compounds produced in the method according to the inventionare themselves steroid hormones and can be used for therapeuticalpurposes.

The compounds produced, such as, for example, ergosta-5,7-dienol orhydrocortisone, can furthermore be used for preparing steroid hormonesor for the synthesis of active ingredients with a potentantiinflammatory, abortive or antiproliferative activity viabiotransformation, chemical synthesis or biotechnological production.

In the method according to the invention for the production ofergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites the step of culturing the genetically modified organisms,hereinbelow also referred to as transgenic organisms, is preferablyfollowed by harvesting of the organisms and isolation ofergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites from the organisms.

The organisms are harvested in a manner known per se to suit theorganism in question. Microorganisms such as bacteria, mosses, yeastsand fungi or plant cells which are grown in liquid nutrient media byfermentation can be separated for example by centrifugation, decantingor filtration.

Ergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites from the harvested biomass are isolated jointly orseparately for each compound in a manner known per se, for example byextraction and, if appropriate, further chemical or physicalpurification processes such as, for example, precipitation methods,crystallography, thermal separation methods like rectification methodsor physical separation methods such as, for example, chromatography.

The invention furthermore relates to a method for generating agenetically modified organism in which, starting from a startingorganism, the Δ22-desaturase activity is reduced and the HMG-CoAreductase activity is increased and at least one of the activitiesselected from the group consisting of lanosterol C14-demethylaseactivity, squalene epoxidase activity and squalene synthetase activityis increased.

The methods for deleting the target locus Δ22-desaturase gene havealready been detailed above.

The transgenic organisms, in particular yeasts, can preferably begenerated by transforming the starting organisms, in particular yeasts,with a nucleic acid construct comprising at least one nucleic acidencoding an HMG-CoA reductase and comprising at least one nucleic acidselected from the group consisting of nucleic acids encoding alanosterol C14-demethylase, nucleic acids encoding a squalene epoxidaseand nucleic acids encoding a squalene synthetase, which nucleic acidsare linked operably to one or more regulatory signals which ensure thetranscription and translation in the organisms. In this embodiment, thetransgenic organisms are generated using a nucleic acid construct.

Nucleic acid constructs which can be used for this purpose are thosewhich, in addition to the expression cassettes described hereinbelow andcomprising promoter, coding sequence and, if appropriate, terminator,and in addition to a selection marker described hereinbelow, comprise,at their 3′ and 5′ ends, nucleic acid sequences which are identical tonucleic acid sequences at the beginning and at the end of the gene to bedeleted.

However, the transgenic organisms may also preferably be generated bytransforming the starting organisms, in particular yeasts, with acombination of nucleic acid constructs comprising nucleic acidconstructs comprising at least one nucleic acid encoding an HMG-CoAreductase and comprising nucleic acid constructs or a combination ofnucleic acid constructs comprising at least one nucleic acid selectedfrom the group consisting of nucleic acids encoding a lanosterolC14-demethylase, nucleic acids encoding a squalene epoxidase and nucleicacids encoding a squalene synthetase and which are in each case linkedoperably to one or more regulatory signals which ensure thetranscription and translation in organisms.

In this embodiment, the transgenic organisms are generated usingindividual nucleic acid constructs or a combination of nucleic acidconstructs.

Nucleic acid constructs in which the coding nucleic acid sequence islinked operably to one or more regulatory signals which ensure thetranscription and translation in organisms, in particular in yeasts, arehereinbelow also referred to as expression cassettes.

Nucleic acid constructs comprising this expression cassette are, forexample, vectors or plasmids.

The regulatory signals preferably comprise one or more promoters whichensure the transcription and translation in organisms, in particular inyeasts.

The expression cassettes comprise regulatory signals, that is to sayregulatory nucleic acid sequences which control the expression of thecoding sequence in the host cell. In accordance with a preferredembodiment, an expression cassette encompasses a promoter upstream, i.e.at the 5′ end of the coding sequence, and a terminator downstream, i.e.at the 3′ end, and, if appropriate, further regulatory elements whichare linked operably to the interposed coding sequence for at least oneof the above-described genes.

Operable linkage is understood as meaning the sequential arrangement ofpromoter, coding sequence, if appropriate terminator and if appropriatefurther regulatory elements in such a way that each of the regulatoryelements can fulfill its intended function upon expression of the codingsequence.

By way of example, the preferred nucleic acid constructs, expressioncassettes and plasmids for yeasts and fungi and methods for generatingtransgenic yeasts and the transgenic yeasts themselves are described inthe following text.

A suitable promoter for the expression cassette is, in principle, anypromoter which is capable of controlling the expression of foreign genesin organisms, in particular in yeasts.

A promoter which is preferably used is, in particular, a promoter whichis subject to reduced regulation in yeast, such as, for example, themiddle ADH promoter.

This promoter fragment of the ADH12s promoter, hereinbelow also referredto as ADH1, shows approximately constitutive expression (Ruohonen L,Penttila M, Keranen S. (1991) Optimization of Bacillus alpha-amylaseproduction by Saccharomyces cerevisiae. Yeast. May-June; 7(4):337-462;Lang C, Looman A C. (1995) Efficient expression and secretion ofAspergillus niger RH5344 polygalacturonase in Saccharomyces cerevisiae.Appl Microbiol Biotechnol. December; 44(1-2):147-56.), so thattranscriptional regulation is no longer effected by ergosterolbiosynthesis intermediates.

Further preferred promoters with reduced regulation are constitutivepromoters such as, for example, the yeast TEF1 promoter, the yeast GPDpromoter or the yeast PGK promoter (Mumberg D, Muller R, Funk M. (1995)Yeast vectors for the controlled expression of heterologous proteins indifferent genetic backgrounds. Gene. 1995 Apr. 14; 156(1):119-22; Chen CY, Oppermann H, Hitzeman R A. (1984) Homologous versus heterologous geneexpression in the yeast, Saccharomyces cerevisiae. Nucleic Acids Res.December 11; 12(23):8951-70.).

The expression cassette may also comprise inducible promoters, inparticular chemically inducible promoters, by means of which theexpression, in the organism, of the nucleic acids encoding an HMG-CoAreductase, lanosterol C14-demethylase, squalene epoxidase or squalenesynthetase can be controlled at a particular point in time.

Such promoters such as, for example, the yeast Cupl promoter,(Etcheverry T. (1990) Induced expression using yeast coppermetallothionein promoter. Methods Enzymol. 1990; 185:319-29.), the yeastGal1-10 promoter (Ronicke V, Graulich W, Mumberg D, Muller R, Funk M.(1997) Use of conditional promoters for expression of heterologousproteins in Saccharomyces cerevisiae, Methods Enzymol. 283:313-22) orthe yeast Pho5 promoter (Bajwa W, Rudolph H, Hinnen A. (1987) PHO5upstream sequences confer phosphate control on the constitutive PHO3gene. Yeast. 1987 March; 3(1):33-42) may be used by way of example.

A suitable terminator for the expression cassette is, in principle, anyterminator which is capable of controlling the expression of foreigngenes in organisms, in particular in yeasts.

The yeast tryptophan terminator (TRP1 terminator) is preferred.

An expression cassette is preferably generated by fusing a suitablepromoter to the above-described nucleic acids encoding an HMG-CoAreductase, lanosterol C14-demethylase, squalene epoxidase or squalenesynthetase and, if appropriate, a terminator using customaryrecombination and cloning techniques as are described, for example, inT. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989), in T. J. Silhavy, M. L. Berman and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Greene Publishing Assoc. andWiley-Interscience (1987).

The nucleic acids according to the invention can have been synthesizedor obtained naturally or comprise a mixture of synthetic and naturalnucleic acid components, or else consist of various heterologous genesegments from various organisms.

Preferred are, as described above, synthetic nucleotide sequences withcodons which are preferred by yeasts. These codons which are preferredby yeasts can be determined from codons with the highest proteinfrequency which are expressed in most of the yeast species of interest.

When preparing an expression cassette, various DNA fragments can bemanipulated in order to obtain a nucleotide sequence which expedientlyreads in the correct direction and is equipped with the correct readingframe. Adapters or linkers may be added to the fragments in order tolink the DNA fragments with one another.

The promoter and terminator regions may expediently be provided, in thedirection of transcription, with a linker or polylinker comprising oneor more restriction cleavage sites for the insertion of this sequence.As a rule, the linker has 1 to 10, in most cases 1 to 8, preferably 2 to6, restriction cleavage sites. In general, the linker within theregulatory regions has a size of less than 100 bp, frequently less than60 bp, but at least 5 bp. The promoter may be either native, orhomologous, or else foreign, or heterologous, with respect to the hostorganism. The expression cassette preferably comprises, in the 5′-3′direction of transcription, the promoter, a coding nucleic acid sequenceor a nucleic acid construct and a region for transcriptionaltermination. Various termination regions can be exchanged for oneanother as desired.

Manipulations which provide suitable restriction cleavage sites or whichremove superfluous DNA or restriction cleavage sites may furthermore beemployed. Where insertions, deletions or substitutions such as, forexample, transitions and transversions, are suitable, in vitromutagenesis, primer repair, restriction or ligation may be used.

Suitable manipulations such as, for example, restriction, chewing backor filling in overhangs for blunt ends may provide complementary ends ofthe fragments for the ligation.

The invention furthermore relates to the use of the above-describednucleic acids, the above-described nucleic acid constructs or theabove-described proteins for the generation of transgenic organisms, inparticular yeasts.

These transgenic organisms, in particular yeasts, preferably have anincreased content in ergosta-5,7-dienol and/or its biosyntheticintermediates and/or metabolites in comparison with the wild type.

The invention furthermore relates to the use of the above-describednucleic acids or of the nucleic acid constructs according to theinvention for increasing the content in ergosta-5,7-dienol and/or itsbiosynthetic intermediates and/or metabolites in organisms.

The above-described proteins and nucleic acids can be used for producingergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites in transgenic organisms.

The transfer of foreign genes into the genome of an organism, inparticular of yeast, is referred to as transformation.

Transformation methods which are known per se may be used for thispurpose, in particular in yeasts.

Suitable methods for transforming yeasts are, for example, the LiACmethod as described in Schiestl R H, Gietz R D. (1989) High efficiencytransformation of intact yeast cells using single stranded nucleic acidsas a carrier, Curr Genet. December; 16(5-6):339-46, the electroporationas described in Manivasakam P, Schiestl R H. (1993) High efficiencytransformation of Saccharomyces cerevisiae by electroporation. NucleicAcids Res. September 11; 21(18):4414-5, or the preparation ofprotoplasts as described in Morgan A J. (1983) Yeast strain improvementby protoplast fusion and transformation, Experientia Suppl. 46:155-66.

The construct to be expressed is preferably cloned into a vector, inparticular into plasmids which are suitable for the transformation ofyeasts, such as, for example, the vector systems Yep24 (Naumovski L,Friedberg E C (1982) Molecular cloning of eucaryotic genes required forexcision repair of UV-irradiated DNA: isolation and partialcharacterization of the RAD3 gene of Saccharomyces cerevisiae. JBacteriol October; 152(1):323-31), Yep13 (Broach J R, Strathern J N,Hicks J B. (1979) Transformation in yeast: development of a hybridcloning vector and isolation of the CAN1 gene. Gene. 1979 December;8(1):121-33), the pRS vector series (Centromer and Episomal) (Sikorski RS, Hieter P. (1989) A system of shuttle vectors and yeast host strainsdesigned for efficient manipulation of DNA in Saccharomyces cerevisiae.Genetics. May; 122(1):19-27) and the vector systems YCp19 or pYEXBX.

Accordingly, the invention furthermore relates to vectors, in particularplasmids comprising the above-described nucleic acids, nucleic acidconstructs or expression cassettes.

The invention furthermore relates to a method for the generation ofgenetically modified organisms by functionally inserting, into thestarting organism, an above-described nucleic acid or an above-describednucleic acid construct.

The invention furthermore relates to the genetically modified organisms,where the genetic modification

reduces the Δ22-desaturase activity and

increases the HMG-CoA reductase activity and

increases at least one of the activities selected from the groupconsisting of lanosterol C14-demethylase activity, squalene epoxidaseactivity and squalene synthetase activity

in comparison with the wild type.

In a preferred embodiment, the genetically modified organisms have areduced Δ22-desaturase activity and an increased HMG-CoA reductaseactivity and an increased lanosterol C14-demethylase activity incomparison with the wild type.

In a further preferred embodiment, the genetically modified organismshave a reduced Δ22-desaturase activity and an increased HMG-CoAreductase activity and an increased squalene epoxidase activity incomparison with the wild type.

In a further preferred embodiment, the genetically modified organismshave a reduced Δ22-desaturase activity and an increased HMG-CoAreductase activity and an increased squalene synthetase activity incomparison with the wild type.

In an especially preferred embodiment, the genetically modifiedorganisms have a reduced Δ22-desaturase activity and an increasedHMG-CoA reductase activity and an increased lanosterol C14-demethylaseactivity and an increased squalene epoxidase activity in comparison withthe wild type.

In a further, especially preferred embodiment, the genetically modifiedorganisms have a reduced Δ22-desaturase activity and an increasedHMG-CoA reductase activity and an increased lanosterol C14-demethylaseactivity and an increased squalene synthetase activity in comparisonwith the wild type.

In a further, especially preferred embodiment, the genetically modifiedorganisms have a reduced Δ22-desaturase activity and an increasedHMG-CoA reductase activity and an increased squalene epoxidase activityand an increased squalene synthetase activity in comparison with thewild type.

In a very especially preferred embodiment, the genetically modifiedorganisms have a reduced Δ22-desaturase activity and an increasedHMG-CoA reductase activity and an increased lanosterol C14-demethylaseactivity and an increased squalene epoxidase activity and an increasedsqualene synthetase activity in comparison with the wild type.

As mentioned above, these activities are preferably increased byincreasing independently, in comparison with the wild type, the geneexpression of nucleic acids encoding an HMG-CoA reductase, nucleic acidsencoding a lanosterol C14-demethylase, nucleic acids encoding a squaleneepoxidase or nucleic acids encoding a squalene synthetase.

The furthermore preferred embodiments of the preferred geneticallymodified organisms according to the invention are described hereinabovein the methods.

The above-described genetically modified organisms have an increasedcontent in ergosta-5,7-dienol and/or its biosynthetic intermediatesand/or metabolites in comparison with the wild type.

Accordingly, the invention relates to an above-described geneticallymodified organism, wherein the genetically modified organism has anincreased content in ergosta-5,7-dienol and/or its biosyntheticintermediates and/or metabolites in comparison with the wild type.

Organisms or genetically modified organisms are understood as meaning,in accordance with the invention, for example bacteria, in particularbacteria of the genus Bacillus, Escherichia coli, Lactobacillus spec. orStreptomyces spec.,

for example yeasts, in particular yeasts of the genus Saccharomycescerevisiae, Pichia pastoris or Klyveromyces spec.,

for example fungi, in particular fungi of the genus Aspergillus spec.,Penicillium spec. or Dictyostelium spec.,

and, for example, also insect cell lines which are capable of generatingergosta-5,7-dienol and/or its biosynthetic intermediates and/ormetabolites, either as the wild type or owing to preceding geneticmodification.

Especially preferred organisms or genetically modified organisms areyeasts, in particular of the species Saccharomyces cerevisiae, inparticular the yeast strains Saccharomyces cerevisiae AH22,Saccharomyces cerevisiae GRF, Saccharomyces cerevisiae DBY747 andSaccharomyces cerevisiae BY4741.

Increasing the content in ergosta-5,7-dienol and/or its biosyntheticintermediates and/or metabolites means, for the purposes of the presentinvention, preferably the artificially acquired ability of an increasedbiosynthesis rate of at least one of these compounds mentioned at theoutset in the genetically modified organism in comparison with thenon-genetically-modified organism.

An increased content in ergosta-5,7-dienol and/or its biosyntheticintermediates and/or metabolites in comparison with the wild type isunderstood as meaning in particular increasing the content of at leastone of the abovementioned compounds in the organism by at least 50%, bypreference 100%, more preferably 200%, especially preferably 400% incomparison with the wild type.

The determination of the content in at least one of the abovementionedcompounds is preferably carried out by analytical methods known per seand preferably relates to those compartments of the organism in whichsterols are produced.

The advantage of the present invention in comparison with the prior artis as follows:

The method according to the invention makes it possible to increase thecontent in ergosta-5,7-dienol and/or its biosynthetic intermediatesand/or metabolites in the production organisms.

The invention will now be illustrated by the examples which follow, butis not limited thereto:

I. General Experimental Conditions

1. Restriction

The plasmids (1 to 10 μg) were restricted in 30 μl reactions. To thisend, the DNA was taken up in 24 μl of H₂O and treated with 3 μof thebuffer in question, 1 ml of BSA (bovine serum albumin) and 2 μl ofenzyme. The enzyme concentration was 1 unit/μl or 5 units/μl, dependingon the DNA quantity. In some cases, 1 μl of RNase was also added to thereaction in order to break down the tRNA. The restriction reaction wasincubated for 2 hours at 37° C. The restriction was checked with aminigel.

2. Gel Electrophoreses

The gel electrophoreses were carried out in minigel or wide minigelapparatuses. The minigels (approx. 20 ml, 8 wells) and the wide minigels(50 ml, 15 or 30 wells) consisted of 1% agarose in TAE. The runningbuffer used was 1× TAE. The samples (10 μl) were treated with 3 μl ofstop solution and applied. HindIII-cut I-DNA acted as the standard(bands at: 23.1 kb; 9.4 kb; 6.6 kb; 4.4 kb; 2.3 kb; 2.0 kb; 0.6 kb). Forthe separation, 80 volts were applied for 45 to 60 minutes. Thereafter,the gel was stained in ethidium bromide solution and, under UV light,recorded with the video documentation system INTAS or photographed usingan orange filter.

3. Gel Elution

The desired fragments were isolated by means of gel elution. Therestriction reaction was loaded into several wells of a minigel and run.Only λ-HindIII and a “sacrificial lane” were stained with ethidiumbromide solution and viewed under UV light, and the desired fragment wasmarked. Damage by the ethidium bromide and the UV light to the DNA inthe remaining wells was thus prevented. By placing the stained and theunstained gel slab next to each other, it was possible to excise thedesired fragment from the unstained gel slab with reference to themarker. The agarose section with the fragment to be isolated was placedinto a dialysis tube, sealed with a small amount of TAE buffer withoutair bubbles and placed into the BioRad minigel apparatus. The runningbuffer consisted of 1× TAE, and the voltage applied was 100 V for 40minutes. Thereafter, the polarity of the current was reversed for 2minutes in order to redissolve the DNA which stuck to the dialysis tube.The buffer, of the dialysis tube, which contained the DNA fragments wastransferred into reaction vessels and used for carrying out an ethanolprecipitation. To this end, 1/10 volume of 3M sodium acetate, tRNA (1 μlper 50 μl solution) and 2.5 volumes of ice-cold 96% ethanol were addedto the DNA solution. The reaction was incubated for 30 minutes at −20°C. and then centrifuged for 30 minutes at 4° C. at 12 000 rpm. The DNApellet was dried and taken up in 10 to 50 μl of H₂O (depending on theDNA quantity).

4. Klenow Treatment

The Klenow treatment results in DNA fragment overhangs being filled inso that blunt ends result. The following mixture was pipetted togetherfor each μg of DNA:

-   -   DNA pellet+11 μl H20        -   +1.5 μl 10× Klenow buffer        -   +1 μl 0.1 M DTT        -   +1 μl nucleotide (dNTP 2 mM)        -   25+1 μl Klenow polymerase (1 unit/μl)

The DNA for this purpose should originate from an ethanol precipitationin order to prevent contaminants inhibiting the Klenow polymerase. Themixture was incubated for 30 minutes at 37° C. and the reaction wasstopped by a further 5 minutes at 70° C. The DNA was obtained from themixture by precipitation of ethanol and taken up in 10 μl of H₂O.

5. Ligation

The DNA fragments to be ligated were combined. The final volume of 13.1μl contained approx. 0.5 μl of DNA with a vector:insert ratio of 1:5.The sample was incubated for 45 seconds at 70° C., cooled to roomtemperature (approx. 3 minutes) and then incubated for 10 minutes onice. Thereafter, the ligation buffers were added: 2.6 μl 500 mM trisHClpH 7.5 and 1.3 μl 100 mM MgCl₂, and the mixture was incubated on ice fora further 10 minutes. After addition of 1 μl 500 mM DTT and 1 μl 10 mMATP and another 10 minutes on ice, 1 μl of ligase (1 unit/pl) was added.The whole of the treatment should be carried out as free from vibrationsas possible in order not to separate joined-up DNA ends again. Theligation was carried out overnight at 14° C.

6. E. coli Transformation

Competent Escherichia coli (E. coli) NM522 cells were transformed withthe DNA of the ligation reaction. This was accompanied by a reactionwith 50 μg of the pScL3 plasmid as positive control and a reactionwithout DNA as zero control. For each transformation reaction, 100 μl of8% PEG solution, 10 μl of DNA and 200 μl of competent cells (E. coliNM522) were pipetted into a tabletop centrifuge tube. The reactions wereplaced on ice for 30 minutes and shaken occasionally.

They were then given the thermal shock treatment: 1 minute at 42° C. Forthe regeneration, 1 ml of LB medium was added to the cells and themixtures were incubated for 90 minutes at 37° C. on a shaker. 100 μlportions of the undiluted reactions, of a 1:10 dilution and of a 1:100dilution were plated onto LB+ ampicillin plates and incubated overnightat 37° C.

7. Plasmid Isolation from E. coli (Miniprep)

E. coli colonies were grown overnight in 1.5 ml of LB+ ampicillin mediumin tabletop centrifuge tubes at 37° C. and 120 rpm. On the next day, thecells were centrifuged for 5 minutes at 5000 rpm and 4° C. and thepellet was taken up in 50 μl of TE buffer. Each reaction was treatedwith 100 μl of 0.2 N NaOH, 1% SDS solution, mixed and placed on ice for5 minutes (cell lysis). Thereafter, 400 μl of sodium acetate/NaClsolution (230 μl of H₂O, 130 μl of 3 M sodium acetate, 40 μl of 5M NaCl)were added, and the reaction was mixed and placed on ice for a further15 minutes (protein precipitation). After centrifugation for 15 minutesat 11 000 rpm, the supernatant, which contains the plasmid DNA, wastransferred into an Eppendorf tube. If the supernatant was not entirelyclear, it was recentrifuged. The supernatant was treated with 360 μl ofice-cold isopropanol and incubated for 30 minutes at −20° C. (DNAprecipitation). The DNA was centrifuged off (15 min, 12 000 rpm, 4° C.),the supernatant was discarded, and the pellet was washed in 100 μl ofice-cold 96% ethanol, incubated for 15 minutes at −20° C. andrecentrifuged (15 min, 12 000 rpm, 4° C.). The pellet was dried in aSpeed Vac apparatus and then taken up in 100 μl of H₂O. The plasmid DNAwas characterized by restriction analysis. To this end, 10 μl of eachreaction were restricted and separated by gel electrophoresis in a wideminigel (see above).

8. Plasmid Preparation from E. coli (Maxiprep)

In order to isolate larger amounts of plasmid DNA, the maxiprep methodwas carried out. Two flasks containing 100 ml of LB+ ampicillin mediumwere inoculated with a colony or with 100 μl of a frozen culturecontaining the plasmid to be isolated and incubated overnight at 37° C.and 120 rpm. On the next day, the culture (200 ml) was transferred intoa GSA beaker and centrifuged for 10 minutes at 4000 rpm (2600×g). Thecell pellet was taken up in 6 ml of TE buffer. To digest the cell wall,1.2 ml of lysozyme solution (20 mg/ml TE buffer) were added and themixture was incubated for 10 minutes at room temperature. The cells weresubsequently lysed with 12 ml of 0.2 N NaOH, 1% SDS solution and afurther 5 minutes' incubation at room temperature; The proteins wereprecipitated by addition of 9 ml of cold 3 M sodium acetate solution (pH4.8) and 15 minutes' incubation on ice. After the centrifugation (GSA:13 000 rpm (27 500×g), 20 min, 4° C.), the supernatant, which containedthe DNA, was transferred into a fresh GSA beaker and the DNA wasprecipitated with 15 ml of ice-cold isopropanol and 30 minutes'incubation at −20° C. The DNA pellet was washed in 5 ml of ice-coldethanol and dried in the air (approx. 30-60 min). It was then taken upin 1 ml of H₂O. The plasmid was verified by restriction analysis. Theconcentration was determined by applying dilutions to a minigel. Amicrodialysis (pore size 0.025 μm) was carried out for 30-60 minutes inorder to reduce the salt content.

9. Yeast Transformation

A preculture of the strain Saccharomyces cerevisiae AH22 was establishedfor the yeast transformation. A flask containing 20 ml of YE medium wasinoculated with 100 μl of the frozen culture and incubated overnight at28° C. and 120 rpm. The main culture was carried out under identicalconditions in flasks containing 100 ml of YE medium which had beeninoculated with 10 μl, 20 μl or 50 μl of the preculture.

9.1 Generation of Competent Cells

On the next day, the flasks were counted using a hematocytometer and theflask with a cell concentration of 3-5×10⁷ cells/ml was chosen for thefollowing procedure. The cells were harvested by centrifugation (GSA:5000 rpm (4000×g) 10 min). The cell pellet was taken up in 10 ml of TEbuffer and divided between two tabletop centrifuge tubes (5 ml each).The cells were centrifuged off for 3 minutes at 6000 rpm and washedtwice with in each case 5 ml of TE buffer. The cell pellet wassubsequently taken up in 330 μl of lithium acetate buffer per 10⁹ cells,transferred into a sterile 50 ml Erlenmeyer flask and shaken for onehour at 28° C. The cells were thus competent for the transformation.

9.2 Transformation

For each transformation reaction, 15 μl of herring sperm DNA (10 mg/ml),10 μl of DNA to be transformed (approx 0.5 μg) and 330 μl of competentcells were pipetted into a tabletop centrifuge tube and incubated for 30minutes at 28° C. (without shaking). Thereafter, 700 μl 50% PEG 6000were added and the reactions were incubated for a further hour at 28° C.without shaking. This was followed by 5 minutes' heat shock treatment at42° C. 100 μl of the suspension were plated onto selection medium (YNB,Difco) in order to select for leucine prototrophism. In the case ofselection of G418 resistance, the cells are regenerated following theheat shock treatment (see 9.3, regeneration phase).

9.3 Regeneration Phase

Since the selection marker is the resistance to G418, the cells requiredtime for expressing the resistance gene. The transformation reactionswere treated with 4 ml of YE medium and incubated overnight at 28° C. ona shaker (120 rpm). On the next day, the cells were centrifuged off(6000 rpm, 3 min), taken up in 1 ml of YE medium, and 100 μl or 200 μlof this were plated onto YE+G418 plates. The plates were incubated forseveral days at 28° C.

10. Reaction Conditions for the PCR

The reaction conditions for the polymerase chain reaction must beoptimized for each individual case and are not generally valid for eachprocedure. It is thus possible to vary, inter alia, the amount of DNAemployed, the salt concentrations and the melting point. For ourapproach, it proved suitable to combine the following substances in anEppendorf tube suitable for use in thermocyclers: 5 μl of Super Buffer,8 μl of dNTPs (0.625 μM each), 5′-primer, 3′-primer and 0.2 μg oftemplate DNA, dissolved in such an amount of water that a total volumeof 50 μl for the PCR reaction results, were added to 2 μl (=0.1 U) SuperTaq polymerase. The reaction was centrifuged briefly and covered with adrop of oil. Between 37 and 40 cycles were selected for theamplification.

II. EXAMPLES Example 1

Expression of a Truncated HMG-CoA Reductase in S. cerevisiae GRF

The coding nucleic acid sequence for the expression cassette consistingof ADH-promoter-tHMG-tryptophan-terminator was amplified from the vectorYepH2 (Polakowski et al. (1998) Overexpression of a cytosolichydroxymethylglutaryl-CoA reductase leads to squalene accumulation inyeast. Appl Microbiol Biotechnol. January; 49(1):66-71) by PCR usingstandard methods as detailed above under the general reactionconditions.

The primers used for this purpose are the DNA oligomers AtHT-5′(forward: tHMGNotF: 5′-CTGCGGCCGCATCATGGACCMTTGGTGAAAACTG-3′; SEQ. ID.NO. 11) and AtHT-3′ (reverse: tHMGXhoR:5′-MCTCGAGAGACACATGGTGCTGTTGTGCTTC-3′; SEQ. ID. No. 12).

The resulting DNA fragment was first treated with Klenow and then clonedblunt-ended into the vector, pUG6 into the EcoRV cleavage site, givingrise to the vector pUG6-tHMG (FIG. 1).

Following the isolation of the plasmid, an extended fragment wasamplified from the vector pUG-tHMG by means of PCR so that the resultingfragment consists of the following components:loxP-kanMX-ADH-promoter-tHMG-tryptophan-terminator-loxP. The primerschosen were oligonucleotide sequences which, at the 5′ and 3′ overhangs,comprise the 5′ or the 3′ sequence of the URA3 gene, respectively, andin the annealing region the sequences of the loxP regions 5′ and 3′ ofthe vector pUG-tHMG. This ensures that firstly the entire fragmentincluding KanR and tHMG is amplified and secondly that this fragment cansubsequently be transformed into yeast and the entire fragmentintegrates into the yeast URA3 gene locus by homologous recombination.

The selection marker used is the resistance to G418. The resultingstrain S. cerevisiae GRF-tH1ura3 is Uracil-auxotrophic and contains acopy of the gene tHMG under the control of the ADH promoter andtryptophan terminator.

In order to subsequently remove the resistance to G418 again, theresulting yeast strain is transformed with the cre recombinase vectorpSH47 (Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann J H. (1996)A new efficient gene disruption cassette for repeated use in buddingyeast. Nucleic Acids Res. July 1; 24(13):2519-24.). Owing to thisvector, the cre recombinase is expressed in the yeast, and, as aconsequence, the sequence region within the two loxP sequencesrecombines out of the gene. The result is that only one of the two loxPsequences and the ADH-tHMG-TRP cassette are retained in the URA3 genelocus. As a consequence, the yeast strain loses the G418 resistanceagain and is thus suitable for integrating further genes into the yeaststrain by means of this cre-lox system or removing them, respectively.The vector pSH47 can now be removed again by counterselection on YNBagar plates supplemented with Uracil (20 mg/l) and FOA (5-fluorooroticacid) (1 g/l). To this end, the cells which bear this plasmid must firstbe cultured under nonselective conditions and subsequently be grown onFOA-containing selective plates. Only those cells which are not capableof synthesizing Uracil themselves are capable of growing under theseconditions. In the present case, these are cells which no longer containplasmid (pSH47).

The yeast strain GRFtH 1 ura3 and the original strain GRF were culturedfor 48 hours in WMXIII medium at 28° C. and 160 rpm in a culture volumeof 20 ml. 500 μl of this preculture were subsequently transferred into a50 ml main culture of the same medium and cultured for 4 days at 28° C.and 160 rpm in a baffle flask.

The sterols were extracted after 4 days following the method asdescribed in Parks L W, Bottema C D, Rodriguez R J, Lewis T A. (1985)Yeast sterols: yeast mutants as tools for the study of sterolmetabolism. Methods Enzymol. 1985; 111:333-46 and analyzed by gaschromatography. This gives the data listed in table 1. The percentagesare based on the yeast dry weight.

TABLE 1 Sterol content [peak area/gDM] S. cerevisiae GRFtH1ura3 S.cerevisiae GRF Squalene 9.93 0.1 Lanosterol 0.83 0.31 Zymosterol 1.181.07 Fecosterol 1.10 0.64 Episterol/ergosta-5,7- 1.04 0.72 dienolDimethyl- 0.34 0.13 zymosterol

Example 2

Expression of ERG1 in S. cerevisiae GRFtH1ura3 with SimultaneousDeletion of ERG5; Generation of GRFtH1ura3ERG1erg5

Example 2.1

Generation of the Integration Vector pUG6-ERG1

The DNA sequence for the cassette consisting ofADH-promoter-ERG1-tryptophan-terminator was isolated from the vectorpFlat3-ERG1 by restriction with the enzymes Nhel and Bsp68l(Nrul) usingstandard methods. The resulting DNA fragment was treated with Klenow andthen cloned blunt-ended into the vector pUG6 into the EcoRV cleavagesite, giving rise to the vector pUG6-ERG1 (FIG. 2).

Example 2.2.

Integrative Transformations

Following the isolation of the plasmid, an extended fragment wasamplified from the vector pUG6-ERG1 by means of PCR so that theresulting fragment consists of the following components:loxP-kanMX-loxP-ADH1-Pr.-ERG1-Trp-Term. The primers used wereoligonucleotide sequences which contain, in the annealing region, thesequences beyond the cassette to be amplified, of the vector pUG6-ERG1,and at the 5′ and 3′ overhangs the 5′ or the 3′ sequence of theintegration locus ERG5, respectively. This ensures that firstly theentire fragment including KanR and the target gene ERG1 is amplified andsecondly that this fragment can subsequently be transformed into yeastand integrates into the yeast target gene locus ERG5 by homologousrecombination. The following primers were used for this purpose:

ERG5-Crelox-5′ (SEQ ID NO: 13): 5′-ATGAGTTCTG TCGCAGAAAA TATAATACAACATGCCACTC CCAGCTGAAGCTTCGTACGC-3′ and ERG5-Crelox-3′ (SEQ ID NO: 14):5′-TTATTCGAAG ACTTCTCCAG TAATTGGGTC TCTCTTTTTG GCATAGGCCA CTAGTGGATCTG-3′

The selection marker used is the resistance to geneticin (G418). Theresulting strain contains one copy of the target gene ERG1 under thecontrol of the ADH1 promoter and the tryptophan terminator. Byintegration of the gene it is simultaneously possible to delete thecorresponding gene ERG5 of the target locus. In order to subsequentlyremove the resistance to G418 again, the resulting yeast strain istransformed with the cre recombinase vector pSH47. Owing to this vector,the cre recombinase is expressed in the yeast, and, as a consequence,the sequence region within the two loxP sequences recombines out of thegene, the result of which is that only one of the two loxP sequences andthe cassette consisting of ADH1-prom.-ERG1-TRP1-term. are retained inthe target locus ERG5. As a consequence, the yeast strain loses the G418resistance again. The vector pSH47 can now be removed selectively bycultivation on FOA medium.

The resulting yeast strain GRFtH1ura3ERG1erg5 was cultured for 48 hoursin WMVII medium at 28° C. and 160 rpm in a culture volume of 20 ml. 500μl of this preculture were subsequently transferred into a 50 ml mainculture of the same medium and cultured for 3 days at 28° C. and 160 rpmin a baffle flask.

The sterols were extracted after 4 days following the method asdescribed in Parks L W, Bottema C D, Rodriguez R J, Lewis T A. (1985)Yeast sterols: yeast mutants as tools for the study of sterolmetabolism. Methods Enzymol. 1985; 111:333-46 and analyzed by gaschromatography. This gives the data listed in table 2. The percentagesare based on the yeast dry weight.

TABLE 2 Sterol content [peak S. cerevisiae area/gDM] GRFtH1ura3ERG1erg5S. cerevisiae GRF Squalene 8.1 0.1 Lanosterol 2.42 0.31 Zymosterol 1.351.07 Fecosterol 2.01 0.64 Episterol/ergosta-5,7- 12.21 0.72 dienolDimethyl- 1.02 0.13 zymosterol

Comparative Example 1

Deletion of ERG5 in S. cerevisiae GRFtH1ura3; Generation ofGRFtH1ura3erg5

The deletion of ERG5 in S. cerevisiae GRFtH1ura3 was carried outanalogously to example 2. In order to delete only the ERG5 gene, thesame method was used, but the vector pUG6 was employed instead of thevector pUG6-ERG1. This vector pUG6 contains no cassette consisting ofADH-prom-ERG1-Trp-term. By using this vector, it is possible to deleteone gene, in this case the gene ERG5.

The resulting yeast strain GRFtH1 ura3erg5 was cultured for 48 hours inWMVII medium at 28° C. and 160 rpm in a culture volume of 20 ml. 500 μlof this preculture were subsequently transferred into a 50 ml mainculture of the same medium and cultured for 3 days at 28° C. and 160 rpmin a baffle flask.

The sterols were extracted after 4 days following the method asdescribed in Parks L W, Bottema C D, Rodriguez R J, Lewis T A. (1985)Yeast sterols: yeast mutants as tools for the study of sterolmetabolism. Methods Enzymol. 1985; 111:333-46 and analyzed by gaschromatography. This gives the data listed in table 3. The percentagesare based on the yeast dry weight.

TABLE 3 GRFtH1ura3erg5 Sterol content GRFtH1ura3ERG1erg5 (Comparative[peak area/g DM] (Example 2) example) Squalene 8.1 13.18 Lanosterol 2.420.78 Zymosterol 1.35 0.10 Fecosterol 2.01 1.03 Episterol/ergosta-5,7-12.21 8.98 dienol 4,4-Dimethylzymosterol 1.02 0.21

1. A method for the production of ergosta-5,7-dienol comprisingculturing a genetically modified yeast organism, wherein the geneticmodification reduces the Δ22-desaturase activity consisting of theenzymatic activity of Δ22-desaturase having the amino acid sequence ofSEQ ID.NO: 2 and increases the HMG-CoA reductase activity consisting ofthe enzymatic activity of HMG-CoA reductase having the amino acidsequence of SEQ ID.NO: 4 and increases squalene epoxidase activityconsisting of the enzymatic activity of squalene epoxidase having theamino acid sequence of SEQ ID.NO: 8 in comparison with the wild type. 2.The method as claimed in claim 1, wherein, in order to reduce theΔ22-desaturase activity, the gene expression of a nucleic acid encodinga Δ22-desaturase is reduced in comparison with the wild type organism.3. The method as claimed in claim 2, wherein an organism without afunctional Δ22-desaturase gene is used.
 4. The method as claimed inclaim 1, wherein, in order to increase the HMG-CoA reductase activity,the gene expression of a nucleic acid encoding an HMG-CoA reductase isincreased in comparison with the wild type organism.
 5. The method asclaimed in claim 4, wherein, in order to increase gene expression, anucleic acid construct comprising a nucleic acid encoding an HMG-CoAreductase is introduced into the organism and whose expression in theorganism is subject to reduced regulation in comparison with the wildtype organism.
 6. The method as claimed in claim 5, wherein the nucleicacid construct comprises a promoter which, in the organism, is subjectto reduced regulation in comparison with the wild-type promoter.
 7. Themethod as claimed in claim 6, wherein the nucleic acid encoding anHMG-CoA reductase is a nucleic acid whose expression in the organism issubject to reduced regulation in comparison with the homologous,orthologous nucleic acid.
 8. The method as claimed in claim 7, whereinthe nucleic acid encoding an HMG-CoA reductase is a nucleic acid whichencodes the catalytic region of HMG-CoA reductase.
 9. The method asclaimed in claim 8, wherein the nucleic acids introduced are nucleicacids encoding proteins comprising the amino acid sequence SEQ. ID. NO.4.
 10. The method as claimed in claim 9, wherein a nucleic acidcomprising the sequence SEQ. ID. NO. 3 is introduced.
 11. The method asclaimed in claim 1, wherein, in order to increase the squalene epoxidaseactivity, the gene expression of a nucleic acid encoding a squaleneepoxidase is increased in comparison with the wild type organism. 12.The method as claimed in claim 11, wherein, in order to increase geneexpression, one or more nucleic acids encoding a squalene epoxidase areintroduced into the organism.
 13. The method as claimed in claim 12,wherein the nucleic acids introduced are nucleic acids encoding proteinscomprising the amino acid sequence SEQ. ID. NO.
 8. 14. The method asclaimed in claim 13, wherein a nucleic acid comprising the sequence SEQ.ID. NO. 7 is introduced.